Metabolic profiling as a means of characterizing plant-associated microbial communities

Abstract Microbial communities from leaf and root habitats associated with sugar beet ( Beta vulgaris ) were characterized according to their capacity to metabolize a range of 95 sole carbon sources available in a commercial assay, GN-BIOLOG MicroPlates. Metabolic profiling was assessed as a method for evaluating perturbation of microbial communities of glasshouse-grown sugar beet inoculated with a genetically modified microorganism. This technique has allowed microbial communities, colonising the immature leaves of treated and untreated plants to be differentiated, although no differences were observed when plants inoculated with genetically modified microorganisms and unmodified inoculated plants were compared. As plants developed and differentiated, the carbon utilization patterns observed allowed communities to be grouped according to the habitat from which they were isolated, irrespective of treatment. These studies demonstrate that the genetically modified microorganism, introduced as a seed dressing colonised developing immature tissue throughout the 231-day study but did not disrupt the natural succession of microbial communities in glasshouse-grown sugar beet plants.     

遗传重组微生物的环境监测

遗传重组微生物 (GMM)的环境监测作为生物安全性评价的重要内容之一,正越来越得到广大科研工作者的密切关注。综述了目前遗传工程菌和重组DNA的环境监测的主要方法。一是基于培养的方法,包括利用选择性培养技术,报告基因,基因探针和PCR,免疫监测等;一是基于免培养的方法,包括显微镜观测法,荧光原位杂交技术 (FISH),基于直接提取微生物总DNA的分子生物学方法等。重点介绍了目前常用的几种报告基因和基于直接提取总DNA的DGGE TGGE方法,同时指出我国应加强GMM遗传监控的分子生物学方法的研究及应用。     

Runoff losses of Pseudomonas aureofaciens (lacZY) from soil

Abstract: Leaching of genetically modified microorganisms (GMMs) through soil profiles is generally not a significant concern, since GMMs typically remain near the soil surface following application. The presence of high numbers of GMMs at the soil surface, however, suggests that losses via runoff may occur. Traditional methods of plating nonlabeled bacteria lack precision and are thus seldom used to monitor runoff losses. To examine whether lac ZY, a common genetic marker, could be used to evaluate bacterial runoff from soil, a lac ZY⁺ strain of Pseudomonas aureofaciens 3732 RN-L11 was used at three different pH levels, with and without wheat ( Triticum aestivum L.) cover in a greenhouse experiment. Twelve times over a 245 day period, soils were subjected to simulated rainfall of 84 mm h⁻¹ for a 15 min duration. Runoff losses and survival were quantified at each time. Pseudomonas aureofaciens survived for the longest period at a soil pH of 7; survival was reduced at lower pHs. The number of cells in runoff were usually related to the number of cells surviving in the soil. When high soil populations were present, runoff losses often exceeded 10¹⁰ cfu event⁻¹. When the soil population declined to low or undetectable levels, the runoff contained fewer organisms. Runoff losses of 10⁸ cfu event⁻¹, however, were observed during one runoff event even when the soil population was below the detection limit. This study indicates that lac ZY is an effective marker, and that runoff of GMMs may be an important mechanism for movement to nontarget environments.     

A statistical approach to quantification of genetically modified organisms (GMO) using frequency distributions

Background

According to Regulation (EU) No 619/2011, trace amounts of non-authorised genetically modified organisms (GMO) in feed are tolerated within the EU if certain prerequisites are met. Tolerable traces must not exceed the so-called ‘minimum required performance limit’ (MRPL), which was defined according to the mentioned regulation to correspond to 0.1% mass fraction per ingredient. Therefore, not yet authorised GMO (and some GMO whose approvals have expired) have to be quantified at very low level following the qualitative detection in genomic DNA extracted from feed samples. As the results of quantitative analysis can imply severe legal and financial consequences for producers or distributors of feed, the quantification results need to be utterly reliable.

Results

We developed a statistical approach to investigate the experimental measurement variability within one 96-well PCR plate. This approach visualises the frequency distribution as zygosity-corrected relative content of genetically modified material resulting from different combinations of transgene and reference gene Cq values. One application of it is the simulation of the consequences of varying parameters on measurement results. Parameters could be for example replicate numbers or baseline and threshold settings, measurement results could be for example median (class) and relative standard deviation (RSD). All calculations can be done using the built-in functions of Excel without any need for programming. The developed Excel spreadsheets are available (see section ‘Availability of supporting data’ for details). In most cases, the combination of four PCR replicates for each of the two DNA isolations already resulted in a relative standard deviation of 15% or less.

Conclusions

The aims of the study are scientifically based suggestions for minimisation of uncertainty of measurement especially in —but not limited to— the field of GMO quantification at low concentration levels. Four PCR replicates for each of the two DNA isolations seem to be a reasonable minimum number to narrow down the possible spread of results.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-014-0407-x) contains supplementary material, which is available to authorized users.     

Use of a novel nonantibiotic triple marker gene cassette to monitor high survival of Pseudomonas fluorescens SBW25 on winter wheat in the field

Pseudomonas fluorescens SBW25 was tagged with a triple marker gene cassette containing gfp, encoding green fluorescent protein; luxAB, encoding luciferase; and telABkilA, encoding tellurite resistance, and the tagged strain was monitored in the first Swedish field release of a genetically modified microorganism (GMM). The cells were inoculated onto winter wheat seeds and the GMM cells (SBW25:tgl) were monitored in the field from September 2005 to May 2006 using plating, luminometry and microscopic analyses. Cell numbers were high on all sampling occasions and metabolically active cells were detected on all plant parts. Field results were similar to those obtained in a parallel phytotron study, although the amount of SBW25:tgl detected on shoots was significantly higher in the phytotron than in the field. After winter, cell counts were 100-fold higher on the roots and root-associated soil compared with prewinter measurements, although the cells had a lower relative metabolic activity. The wheat seeds were naturally infested with Microdochium nivale, but no treatment resulted in reduction of disease symptoms. No SWB25:tgl cells were ever found in bulk soil or uninoculated plants. The Swedish field trial results complement and contrast with prior field studies performed with the same parent organism in the United Kingdom under different soil, plant and climatic conditions.     

The long and winding road from the research laboratory to industrial applications of lactic acid bacteria

Research innovations are constantly occurring in universities, research institutions and industrial research laboratories. These are reported in the scientific literature and presented to the scientific community in various congresses and symposia as well as through direct contacts and collaborations. Conversion of these research results to industrially useful innovations is, however, considerably more complex than generally appreciated. The long and winding road from the research laboratory to industrial applications will be illustrated with two recent examples from Chr. Hansen A/S: the implementation in industrial scale of a new production technology based on respiration by Lactococcus lactis and the introduction to the market of L. lactis strains constructed using recombinant DNA technology.     

Demonstration of tra+ plasmid activity in bacteria indigenous to the phyllosphere of sugar beet; gene transfer to a recombinant pseudomonad

Abstract The presence of transfer proficient plasmids in bacteria isolated from the leaves of sugar beet ( Beta vulgaris L.) was studied. Of 435 bacteria sampled 79 (18%) contained plasmids. Pseudomonads (30%), Erwinia (12%) and Klebsiella (9%) were the largest populations sampled of which 22%, 33% and 29%, respectively, contained plasmids. The ability of these plasmids to self-transfer or mediate the mobilization of the tra⁻ mob⁺ broad host range IncQ plasmid R300B was determined. R300B was maintained in 61/79 natural plasmid containing isolates, the Gram positive isolates could not support R300B. Pseudomonas aureofaciens SBW25, isolated from sugar beet leaves, was chromosomally marked with a tetracycline resistance gene and used as a recipient (SBW25ETc). Five isolates of Erwinia herbicola and one of Erwinia salicis containing natural plasmids were able to mobilize R300B into the recombinant, SBW25ETc. These mobolizing ( tra⁺ ) plasmids were not maintained in transconjugant SBW25 cells. Analysis of the fragment patterns of Pst I digested plasmid DNA demonstrated that four (pSB139, pSB140, pSB142, pSB146; 110 kb) were identical, one (pSB153; 65 kb) was common to a subset of fragments in these four and another (pSB169; 100 kb) was unique. Other natural isolates were able to transfer copper resistance ( Erwinia rhapontici , 2 strains) or mercury resistance ( Pseudomonas fluorescens SBW340) to a rifampicin resistant recipient Pseudomonas putida UWC1 but not to SBW25ETc. These self-transferable plasmids were not able to mobilize R300B. These data demonstrate that the phyllosphere supports indigenous microbial populations which have the capacity to transfer genetic material between bacteria of different genera.     

Ecological risk assessment of genetically modified crops based on cellular automata modeling

The assessment of ecological risk in genetically modified (GM) biological systems is critically important for decision-making and public acceptance. Cellular automata (CA) provide a potential modeling and simulation framework for representing relationships and interspecies interactions both temporally and spatially. In this paper, a simple subsystem contains only four species: crop, target pest, non-target pest and enemy insect, and a three layer arrangement of L × L stochastic cellular automata with a periodic boundary were established. The simulation of this simplified system showed abundant and sufficient complexity in population assembly and densities, suggesting a prospective application in ecological risk assessment of GM crops.     

Distribution of pectic epitopes in cell walls of the sugar beet root

Immunolabelling techniques with antibodies specific to partially methyl-esterified homogalacturonan (JIM5: unesterified residues flanked by methylesterified residues. JIM7: methyl-esterified residues flanked by unesterified residues), a blockwise de-esterified homogalacturonan (2F4), 1,4-galactan (LM5) and 1,5-arabinan (LM6) were used to map the distribution of pectin motifs in cell walls of sugar beet root (Beta vulgaris). PME and alkali treatments of sections were used in conjunction with JIM5-7 and 2F4. The JIM7 epitope was abundant and equally distributed in all cells. In storage parenchyma, the JIM5 epitope was restricted to some cell junctions and the lining of intercellular spaces while in vascular tissues it occurred at cell junctions in some phloem walls and in xylem derivatives. After secondary wall formation, the JIM5 epitope was restricted to inner cell wall regions between secondary thickenings. The 2F4 epitope was not detected without de-esterification treatment. PME treatments prior to the use of 2F4 indicated that HG at cell corners was not acetylated. The LM5 epitope was mainly present in the cambial zone and when present in storage parenchyma, it was restricted to the wall region closest to the plasma membrane. The LM6 epitope was widely distributed throughout primary walls but was more abundant in bundles than in medullar ray tissue and storage parenchyma. These data show that the occurrence of oligosaccharide motifs of pectic polysaccharides are spatially regulated in sugar beet root cell walls and that the spatial patterns vary between cell types suggesting that structural variants of pectic polymers are involved in the modulation of cell wall properties.     

Study of rhizosphere and phyllosphere bacterial community and resistance to bacterial canker in genetically engineered phytochrome A cherry plants

The cherry rootstock 'Colt' line was transformed with a phytochrome A rice gene with the aim of altering light perception. Three transgenic events were chosen because of a modified developmental behavior. When red enriched light was supplied horizontally to stems, the PD3 transgenic line showed an increased rate of phytomer formation associated to a superior rate of plant growth compared to wild type (WT). Under the same light conditions, the PO1 and PA lines were less altered in morphology and development. When far-red enriched light was supplied, all transgenic lines had a reduced rate of growth, with the PD3 line being the most similar to the WT. The influence of the alien gene on root and leaf-associated bacteria was studied for a duration of 1 year. Significantly more culturable bacteria were recovered from PA lines than from PO1, PD3 and WT lines. On average, significantly more fluorescent pseudomonads were recovered from the rhizosphere of PA and PO1 lines than from PD3 and WT. No significant differences were detected in the number of bacteria recovered from the phyllosphere of transgenic and WT plant lines. A total of 143 Pseudomonas fluorescens strains isolated from rhizosphere of transgenic and WT lines were tested for their antagonistic activity against Phytophthora nicotianae and differences between bacteria derived from transgenic and WT were not detected. Fluorescent pseudomonads strains isolated from phyllosphere of PA and PO1 lines showed antagonistic activity against P. syringae pv. syringae, whereas no difference among the transgenic and WT lines was detected when fluorescent Pseudomonas strains were tested against P. syringae pv. mors-prunorum. Pathogenicity tests were conducted on rooted and micropropagated plants with P. s. pv. syringae and P. s. pv. mors-prunorum: in all assays, the PO1 lines were the most tolerant to P. s. pv. Syringae, and the PO1 and PD3 were tolerant to P. s. pv. mors-prunorum.     

厌氧氨氧化菌的代谢途径及其关键酶的研究进展

作为新型生物脱氮工艺的代表,厌氧氨氧化反应的代谢功能菌——厌氧氨氧化(Anammox)菌也逐渐成为研究的热点。本文介绍了10种Anammox菌的发现过程,阐述了Anammox菌的关键酶的研究进展。通过对Candidatus"Brocadia anammoxidans"和Candidatus"Kuenenia stuttgartiensis"菌的代谢途径的分析和推理,综述了化学计量模型、生化反应模型和能量代谢模型,并提出了一种可能存在的基于关键酶的厌氧氨氧化菌微界面代谢新途径。     

Carbon source utilization of soil extracted microorganisms as a tool to detect the effects of soil supplemented with genetically engineered and non-engineered Corynebacterium glutamicum and a recombinant peptide at the community level

Abstract: Substrate utilization of microbial cells extracted from soil with a 0.85% aqueous sodium chloride solution, was determined to estimate effects on soil microorganisms at the community level with microtiter plates (Biolog GN®) containing 95 different sources of organic carbon. A consistent pattern of utilized substrates was obtained after 24 h of microtiter plate incubation at 28°C. The absorbance values (OD₅₉₀) obtained from a microtiter plate reader after background correction were transformed by using the average absorbance values of oxidized substrates as a threshold to distinguish between well utilized and poorly or non-utilized substrates and thereby reduce variances between replicates. Doubling times of the extracted soil microorganisms in the microtiter plates were tested with 12 substrates and ranged from 1.96 h to 3.23 h, depending on the carbon source. The carbon source utilization assay was used to assess the effects of soil inoculation with Corynebacterium glutamicum with and without a genetically engineered plasmid (pUN1; 6.3 kb), which encoded for the synthesis of the mammalian protease inhibiting peptide, aprotinin. Additionally, aprotinin itself was added at two concentrations to soil samples. An identical decrease in the number of carbon sources utilized, especially carbohydrates, occurred upon soil inoculation with both C. glutamicum strains after inoculation with 10⁶ cells g⁻¹ soil. This effect was only detectable during the first three weeks of incubation, as long as cell numbers of C. glutamicum (pUN1) were above 10⁵ cfu g⁻¹. Soil amendment with aprotinin resulted in utilization of additional substrates, most of them carbohydrates. With 0.1 mg aprotinin g⁻¹ soil this stimulation lasted 2 days and with 10 mg g⁻¹ it lasted for 7 days.     

Glyphosate applied to genetically modified herbicide-tolerant sugar beet and 'volunteer' potatoes reduces populations of potato cyst nematodes and the number and size of daughter tubers

Glyphosate, applied early or later or twice to genetically modified glyphosate‐tolerant sugar beet, gave excellent control of planted ‘volunteer’ potatoes growing within the crop compared to conventional herbicide programmes with or without clopyralid. In three out of four trials, this resulted in significant reductions in the numbers of eggs and cysts of potato cyst nematodes (Globodera rostochiensis and G. pallida) where infestations were moderate (23–89 eggs g^?1 soil). In the fourth trial, which had very high initial populations (130 eggs ^?1 soil), none of the herbicide treatments had any significant effect on numbers of nematode eggs or cysts. This was probably due to competition for feeding sites, and the early death of the potatoes in all treatments caused by feeding damage by the nematodes and infection by blight, which prevented the nematodes from completing their life cycle. Glyphosate also significantly reduced the number and size of daughter tubers produced, thus helping to prevent a further volunteer problem in the next crop in the rotation. This was achieved by one or two applications of one chemical compared to 2–5 applications of cocktails of conventional herbicides.     

Assessment of Gene Flow from Genetically Modified Anthracnose-Resistant Chili Pepper (Capsicum annuum L.) to a Conventional Crop

We conducted a 2-year field assessment of the gene flow from genetically modified (GM) chili pepper (Capsicum annuum L.), containing the PepEST (pepper esterase) gene, to a non-GM control line “WT512” and two commercial hybrid cultivars, “Manidda” and “Cheongpung Myeongwol (CM).” After seeds were collected from the pollen-recipient non-GM plants, hybrids between them and the GM peppers were screened by a hygromycin assay. PCR with the targeting hpt gene was performed to confirm the presence of transgenes in hygromycin-resistant seedlings. Out of 7,071 “WT512” seeds and 6,854 “Manidda” seeds collected in 2006, eight and 12 hybrids, respectively, were detected. In 2007, 33 hybrids from 3,456 “WT512” seeds and 50 hybrids from 3,457 “CM” seeds were found. The highest frequency of gene flow, 6.19%, was observed in that 2007 trial. These results suggest that a limited isolation distance would be sufficient to prevent gene flow from GM to conventionally bred chili peppers.     

Eco-efficiency for Pollution Prevention in Small to Medium-Sized Enterprises: A Case from South Korea

A simple method of representing the eco-efficiency (E/E) of a product system has been developed and applied to a pollution prevention program at a small to medium-sized enterprise (SME). Cost-side and environment-side indicators were derived using total cost accounting and life-cycle assessment, respectively. The derived indicators were subsequently normalized to reference values representing the current cost and environmental situation. By combining these normalized indicators, the E/E of a product system can be expressed on a simple graph. The method was applied in a case study carried out at a South Korean SME producing components for electronic equipment such as mobile communication base stations. A silver-plating process was identified as one of the key processes driving a substantial fraction of the total cost and aggregate environmental impact of the product system. Focusing on the key issues identified, a series of alternative processes, including use of a product insulation cover, a sieve-type ancillary electrode, a balanced-uniform plating technique, stream segregation, and noncyanide electroplating, were proposed. The feasibility of these alternatives was validated against product specifications as well as the company's financial and spatial capacity. The potential improvements accruing from these alternatives are presented as a simple graph that can be used by decision makers to readily identify trade-offs between economic and environmental issues.     

Assessment of the food safety issues related to genetically modified foods

International consensus has been reached on the principles regarding evaluation of the food safety of genetically modified plants. The concept of substantial equivalence has been developed as part of a safety evaluation framework, based on the idea that existing foods can serve as a basis for comparing the properties of genetically modified foods with the appropriate counterpart. Application of the concept is not a safety assessment per se, but helps to identify similarities and differences between the existing food and the new product, which are then subject to further toxicological investigation. Substantial equivalence is a starting point in the safety evaluation, rather than an endpoint of the assessment. Consensus on practical application of the principle should be further elaborated. Experiences with the safety testing of newly inserted proteins and of whole genetically modified foods are reviewed, and limitations of current test methodologies are discussed. The development and validation of new profiling methods such as DNA microarray technology, proteomics, and metabolomics for the identification and characterization of unintended effects, which may occur as a result of the genetic modification, is recommended. The assessment of the allergenicity of newly inserted proteins and of marker genes is discussed. An issue that will gain importance in the near future is that of post-marketing surveillance of the foods derived from genetically modified crops. It is concluded, among others that, that application of the principle of substantial equivalence has proven adequate, and that no alternative adequate safety assessment strategies are available.